HLA Typing for the Next Generation Mayor, Neema P; Robinson, James; McWhinnie, Alasdair J M ...
PloS one,
05/2015, Letnik:
10, Številka:
5
Journal Article
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Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the ...phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.
Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and ...transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.
During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives ...neurogenesis. Although significant progress has been made in dissecting the acquisition of ;eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. We show here that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. We find that these transcription factors interact in vitro and provide indirect evidence that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation.
Malignant transformation of struma ovarii is a rare event observed in about 5% of cases. We present here an unusual case that closely simulated highly differentiated follicular carcinoma, an entity ...with a relatively similar appearance to malignant struma ovarii. In our case, peritoneal and systemic dissemination occurred 18 y after excision of struma ovarii, which had been reported as histopathologically benign. There were 3 noteworthy features in the case: the highly functioning nature of the lesions (evidenced by a low level of thyroid-stimulating hormone even without thyroxine suppression); the low to minimal (18)F-FDG avidity of the foci, which reiterates the well-differentiated nature of these lesions, as expected in highly differentiated follicular carcinoma; and prominent involvement of rare sites such as spleen and liver, in addition to usual sites such as lungs, peritoneum, and bilateral adnexae.
To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated ...our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.
► CBFA2T3-GLIS2 is a recurrent fusion gene in pediatric AMKL ► CBFA2T3-GLIS2 AMKL has a distinct expression profile and an inferior outcome ► CBFA2T3-GLIS2 induces BMP signaling and enhanced self-renewal of progenitor cells
We demonstrate that genome sequences approaching finished quality can be generated from short paired reads. Using 36 base (fragment) and 26 base (jumping) reads from five microbial genomes of varied ...GC composition and sizes up to 40 Mb, ALLPATHS2 generated assemblies with long, accurate contigs and scaffolds. Velvet and EULER-SR were less accurate. For example, for Escherichia coli, the fraction of 10-kb stretches that were perfect was 99.8% (ALLPATHS2), 68.7% (Velvet), and 42.1% (EULER-SR).
Aim We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based ...typing (SS-SBT) method. However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT®) DNA sequencer PacBio RS II method to our analysis in order to test whether it might determine the HLA allele sequences in some of the loci with which we previously had difficulties. Here, we report on sequence-based genotyping from the promoter-enhancer region to 3′ UTR of the major HLA genes in the Japanese using the PacBio RS II and Ion PGM NGS systems. Method Forty-six DNA samples were obtained from a reference set used previously to establish the HLA allele frequency data in the Japanese population. The reference samples represented more than 99.5% of the HLA alleles at each of the nine HLA loci. The genomic DNA samples were amplified by long ranged PCR using eleven HLA loci specific primer pairs (A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1). After NGS, consensus sequences were obtained via Long Amplicon analysis such as overlapping, clustering of filtered sub-reads, phasing and removing PCR artifacts. The HLA allele sequences for generating a library of consensus sequences were determined by mapping of sequence reads obtained from Ion PGM sequencer. Results A total of 219 HLA allele sequences (20 A, 40 B, 23 C, 31 DRB1, 36 DQA1, 14 DQB1, 15 DPA1 and 40 DPB1) that covered the promoter-enhancer region to 3’UTR were determined for the 46 samples. The classification of the newly identified SNPs and/or indels revealed at least three non-synonymous substitutions for one B and two DPA1 alleles, although most of the substitutions were observed in intron regions. Conclusion We determined at least 219 Japanese major HLA alleles at the field 4 level by NGS and we expect that this HLA genotyping method of entire HLA gene regions by NGS will help to precisely detect rare, novel and null alleles in population genetic and disease studies.
Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method for the field 4 level of allelic resolution to efficiently detect new and null alleles without phase ...ambiguity by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from the promoter-enhancer region to 3’UTR for 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1 using NGS platforms such as Ion PGM, GS Junior and MiSeq. However, HLA alleles could not be assigned at the field 4 level in some HLA loci such as HLA-DQA1, -DPA1 and -DPB1, because SNP and indel densities to separate both of the phases were much lower than in other HLA loci. In this respect, third generation sequencers such as single molecule, real-time (SMRT®) DNA sequencer PacBio RS II (2.5∼10kb) are expected to solve this current genotyping problem to improve the SS-SBT method. Here we report an accuracy of genotyping results obtained from SMRT® technology.
Ten genomic DNAs (TU1∼TU10) obtained from Japanese were provided for long ranged PCR amplification using eight HLA loci specific primer pairs (A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1). Consensus sequences were obtained via HlaTools/Long Amplicon analysis such as overlapping, clustering of filtered subreads, phasing and removing PCR artifacts.
HLA sequences obtained from 10 pooled amplicon samples applied to SMRT® technology with HlaTools/Long Amplicon analysis were compared to published reference sequences for consensus alleles. A total of 153 amplicon sequences were compared, 117 with the field 4 level sequences and 36 with the field 3 level sequences. At the field 3 level 100% of sequences were identical to reference ones while ∼70% (80/117) of sequences were identical at the field 4 level. Error sequences were mostly localized to intronic single- and di-nucleotide homopolymer regions.
The SMRT® technology is useful for highly accurate DNA typing for all the classical HLA loci.
The aim of this study was to evaluate PacBio sequencing technology for HLA typing of multiple DNA samples using multiplexing library preparation strategies that enable pooling of samples and loci.
...Three multiplexing library preparation strategies were evaluated: Multiplex sequencing using symmetric and asymmetric paired HLA amplification primers versus NGSgo® HLA amplification (GenDx) in combination with symmetric adapter ligation. Barcode-tailed HLA-locus specific amplification primers were used to amplify the full-length HLA class I gene of HLA-A, -B, and -C. For this evaluation test, 8 different 16bp barcode sequences were evaluated in symmetric and asymmetric paired multiplex sequencing. For the asymmetric paired barcodes, different barcodes occur at each end to allow for testing of 28 genomic reference DNA samples. For the symmetric paired barcodes, 8 genomic reference DNA samples were tested for the barcode-tailed amplification primers as well as the barcode-tailed adapters.
Following amplification, intra-sample amplicons and loci were pooled over 3 libraries, keeping the three sequencing strategies separate for analysis. The libraries were run on the PacBio® RS II system, and the resulting sequence reads were aligned to generate a highly accurate consensus and typed using GenDx NGSengine® HLA typing software.
We will present data on the performance of the HLA-locus specific amplification using the symmetric and asymmetric paired barcoded primer sets. We will show the effect of the amplicon pooling on typing accuracy and reliability using the different multiplexing sequencing strategies. We will show the sequencing data of equimolar pools of 3 HLA full-length genes from different heterogeneous reference samples sequenced in a single SMRT® Cell. We will also demonstrate the full-length information (all exons+introns) for allele-level genotyping along with SNP phasing information.
Using PacBio’s barcode strategy, multiple DNA samples can be sequenced for all HLA class I genes in a high-throughput setup to provide quick turn-around of HLA typing results. SMRT Sequencing and analysis using the Long Amplicon Analysis Pipeline can generate long, highly accurate DNA sequences suitable for allele-level resolution HLA typing using the NGSengine HLA typing software.
Evaluation of weather forecasting systems and assessment of existing verification procedures are essential to achieve desirable seamless rainfall prediction. Prediction of wet and dry spells is quite ...useful in agriculture and hydrology but very few attempts have been made so far to resolve the issue using numerical model output. Performance of five state-of-the-art global atmospheric general circulation models and their ensemble mean has been examined in predicting the parameters of wet and dry spells (WSs/DSs) during monsoon period of 2008–2011 over seven subzones of the Indian region. The number of WSs across the region is found to be underestimated, while total duration and rainfall amount of WSs (DSs) overestimated (underestimated). Start of the first WS is late and ends of the last WS early in the model forecast. More uncertainty is noticed in the prediction of DS rainfall and its duration than that of the WS. The percentage area of India under wet conditions (rainfall amount over each grid is more than its daily mean monsoon rainfall) and rainwater over the wet area is overestimated by about 59 and 32 %, respectively, in all models.
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